In vitro research involving horses refers to the study of equine cells, tissues, or biological molecules outside their normal biological context, typically in controlled laboratory environments. This research approach allows scientists to investigate cellular processes, molecular interactions, and the effects of various treatments without the ethical and logistical complexities of in vivo studies. In vitro studies contribute to understanding equine physiology, pathology, and pharmacology by providing insights into cellular responses to pathogens, drugs, and other stimuli. This page compiles peer-reviewed research studies and scholarly articles that explore various in vitro methodologies and their applications in equine science, including cell culture techniques, molecular assays, and drug efficacy testing.
Darlington RW, James C.Darlington, R. W. (St. Jude Children's Research Hospital, Memphis, Tenn.), and C. James. Biological and morphological aspects of the growth of equine abortion virus. J. Bacteriol. 92:250-257. 1966.-The growth of equine abortion virus (EAV) was studied by bioassay and electron microscopy in L-cell monolayer and suspension cultures, and in HeLa and BHK 21/13 cell monolayers. Results of virus assay (plaque-forming units) indicated that production of cell-associated virus (CAV) began at 6 to 9 hr after infection in all of the cell strains used. Virus release occurred 1 to 2 hr later. By 15 to 20 h...
Channing CP.OUR knowledge of the pathways of steroid biosynthesis in the ovary has been gained mainly by incubations of ovaries in vitro1,2. The tissues incubated have contained numerous cell types: granulosa cells, theca interna cells, stromal cells, interstitial cells, and sometimes luteal cells. Possibly such mixtures of two or more different cell types are able to secrete hormones that one cell type cannot secrete by itself3–9. Furthermore, during such incubations in vitro an exchange of precursors and products between different cell types may be facilitated because of breakdown of naturally occurri...
Black JA, Leaf G.1. The effects of cyanogen bromide on horse-heart cytochrome c and horse-heart myoglobin have been investigated. Cytochrome c yielded four fragments, of which two were haemopeptides. The two colourless peptides had amino acid compositions corresponding to those that are expected, on the basis of the sequence proposed for horse-heart cytochrome c by Margoliash, Smith, Kreil & Tuppy (1961), from cleavage at both methionine residues. Of the two haemopeptides, one was isolated and shown to be that derived from cleavage at only one methionine residue, that nearer to the C-terminus of the peptid...
HARDY FM, ARBITER D.Hardy, Frank M. (Fort Detrick, Frederick, Md.), and David Arbiter. Lipid inclusions in L cells associated with Venezuelan equine encephalomyelitis virus infection. J. Bacteriol. 89:1101-1103. 1965.-Venezuelan equine encephalomyelitis (VEE) virus has been shown to induce changes of lipid components within the L cell. Lipid inclusions in the form of dark granular bodies were observed in the L cell after aqueous osmium tetroxide fixation and Sudan black staining. Microscopic examination of cells as early as 8 hr after infection with VEE virus showed an increase in the concentration of these inclu...
ROCKEY JH, KLINMAN NR, KARUSH F.Anti-p-azophenyl-beta-lactoside (Lac) antibody has been isolated from equine antiserum by specific precipitation with Lac-HSA (human serum albumin) and Lac-Hy (hemocyanin). Antibody was separated from antigen by chromatography on DEAE cellulose and zone electrophoresis in solvents containing lactose. Six antigenically distinct immunoglobulins have been identified in purified equine anti-Lac antibody: 7S beta(2A)-globulin, 19S gamma-globulin, a 10S gamma(1) globulin, and three antigenically distinct 7S gamma-globulins. The specific hapten-binding activity of the 7S beta(2A)-antibody and of the ...
RANDALL CC, WALKER BM.Randall, Charles C. (University of Mississippi School of Medicine, Jackson) and Barbara M. Walker. Degradation of deoxyribonucleic acid and alteration of nucleic acid metabolism in suspension cultures of L-M cells infected with equine abortion virus. J. Bacteriol. 86:138-146. 1963.-Metabolic alterations in log-phase suspension cultures infected with equine abortion virus (EAV) were determined in L-M cells simultaneously labeled or prelabeled with H(3)- or C(14)-thymidine. Although infection produced an early stimulation of the uptake of labeled thymidine (TdR) into the acid-soluble fraction of...
Arroyo-Salvo C, Río S, Bogetti ME, Plaza J, Miragaya M, Yaneff A, Davio C, Fissore R, Gervasi MG, Gambini A, Perez-Martinez S.Factors contributing to the limited success of in vitro fertilization in horses remain to be studied. In this work, we elucidated the effect of different essential capacitation media components, bicarbonate, and bovine serum albumin or polyvinyl-alcohol, and the incubation microenvironment on sperm parameters associated with capacitation, acrosome reaction, and their ability to activate oocytes via heterologous intracytoplasmic spermatozoa injection in equine cryopreserved spermatozoa. Methods: Frozen-thawed spermatozoa underwent incubation at different time intervals in either Tyrode's albumi...
Aguila L, Cabrera P, Arias ME, Silva M, Felmer R.Intracytoplasmic sperm injection (ICSI) in horses is currently employed for clinical and commercial uses, but the protocol could be optimized to improve its efficiency. We have hypothesized that destabilization of plasma and acrosomal membranes prior to injection would positively impact the developmental potential of equine zygotes generated by ICSI. This study evaluated effects of the sperm treatment with lysolecithin on plasma and acrosomal membranes and on oocyte activation ability, initially following heterologous ICSI on bovine oocytes and subsequently employing equine oocytes. The effect...
Fuentes F, Contreras MJ, Arroyo-Salvo C, Cabrera P P, Silva M, Merino O, Arias ME, Felmer R.Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro, the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidi...
Silva AH, Lucena JEC, Taran FMP, Cunha DS, Almeida JAT, Lima RS, Dias WS, Santiago JM.This study aimed to compare the digestibility of tropical grasses by horses by the in vivo method using mobile nylon bags with the in vitro digestibility method using horse feces as a source of inoculum. Five horses were used in a 2 × 5 factorial design with randomized blocks featuring two methods (in vivo and in vitro) and five grasses: Tifton 85 hay (Cynodon spp.), sixweeks threeawn grass (Aristida adsencionis, Linn), Alexandergrass (Brachiaria plantaginea (Link) Hitchc.), capim-de-raiz (Chloris orthonoton, Doell), and Sabi grass (Urochloa mosambicensis). No difference (P>0.05) was foun...
Gibb Z, Aitken RJ, Sheridan AR, Holt B, Waugh S, Swegen A.The in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit "classical" features of mPTP; specifically, they are resistant to cyclosporin A-mediated inhibition...
Leemans B, Gadella BM, Marchand JHEAM, Van Soom A, Stout TAE.We recently developed re-differentiated equine oviduct epithelial cell (REOEC) monolayers demonstrating various in vivo morphological characteristics, but lacking secondary ciliation. In this study, we evaluated the effects of fetal bovine serum, reproductive steroid hormones, Wnt- and Notch ligands and inhibitors, and different EOEC seeding densities, in both conventional wells and on microporous membranes, on EOEC morphology and, in particular, secondary ciliation. REOEC monolayers were assessed by confocal microscopy after combined staining of nuclei, cilia and the cytoskeleton. Only Wnt li...
Brodesser DM, Kummer S, Eichberger JA, Schlangen K, Corteggio A, Borzacchiello G, Bertram CA, Brandt S, Pratscher B.The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq re...
Luis-Calero M, Ortiz-Rodríguez JM, Fernández-Hernández P, Muñoz-García CC, Pericuesta E, Gutiérrez-Adán A, Marinaro F, Embade N, Conde R....In vitro embryo production is a highly demanded reproductive technology in horses, which requires the recovery (in vivo or post-mortem) and in vitro maturation (IVM) of oocytes. Oocytes subjected to IVM exhibit poor developmental competence compared to their in vivo counterparts, being this related to a suboptimal composition of commercial maturation media. The objective of this work was to study the effect of different concentrations of secretome obtained from equine preovulatory follicular fluid (FF) on cumulus-oocyte complexes (COCs) during IVM. COCs retrieved in vivo by ovum pick up (OPU) ...
Stedman JM, Lutter JD, Hallman C, Laverty S, Richard H, Santschi EM.High-field MRI of the equine stifle provides high-resolution information about soft tissues that is useful in the diagnosis of stifle lameness. The aim of this prospective anatomic study was to describe the appearance, position, size, and shape of the equine femorotibial ligaments, meniscal ligaments, and menisci using 3 Tesla MRI under extended, extended-loaded, and flexed conditions. Additionally, histologic examination of the collateral and cruciate ligaments (CLs) of a single stifle was performed to compare with MRI images. In extension, mild variations in MRI signal intensity were apparen...
Giraldo A, Koch TG, Madan P, Lepage S, Monteith G, Alizadeh AH, Tran A, Mortagy N, Koenig JB.There is a knowledge gap regarding the effect of extracorporeal shockwave treatment (ESWT) on the stress response and immunomodulatory and anti-inflammatory properties of equine umbilical cord blood mesenchymal stromal cells (CB-MSCs). The objective of this study was to investigate the presence of cellular oxidative stress, inflammatory response, and production of growth factors in CB-MSCs after treatment with ESWT. We hypothesized that CB-MSCs treated with ESWT will experience higher levels of cellular stress and increased production of anti-inflammatory cytokines and growth factors compared ...
Sala-Ayala L, Pytel AT, Stychno K, Cuervo-Arango J.Ovum pick-up (OPU) by transvaginal ultrasound guided follicle aspiration in mares is a common assisted reproductive technique used for oocyte recovery and in vitro production of horse embryos. There has been relatively little research into the factors influencing oocyte recovery in OPU from live mares. The objective of this study was to compare oocyte recovery and morphology of ultrasound-guided follicle puncture and aspiration in live mares and in postmortem excised ovaries, in order to validate an experimental model for research purposes of the efficiency of OPU in mares. Data from OPU perfo...
Froidurot A, Jacotot E, Julliand S, Grimm P, Julliand V.Despite their low quantity and abundance, the cellulolytic bacteria that inhabit the equine large intestine are vital to their host, as they enable the crucial use of forage-based diets. Fibrobacter succinogenes is one of the most important intestinal cellulolytic bacteria. In this study, Fibrobacter sp. HC4, one cellulolytic strain newly isolated from the horse cecum, was characterized for its ability to utilize plant cell wall fibers. Fibrobacter sp. HC4 consumed only cellulose, cellobiose, and glucose and produced succinate and acetate in equal amounts. Among genes coding for CAZymes, 26% o...
Wong YS, Mançanares AC, Navarrete F, Poblete P, Mendez-Pérez L, Cabezas J, Riadi G, Rodríguez-Alvarez L, Castro FO.Mare endometrosis is a major reproductive problem associated with low fertility and is characterized by persistent inflammation, TGFβ-1 signaling, and consequently, extracellular matrix deposition, which compromises endometrial glands. Mesenchymal stem cell-based products (MSCs), such as extracellular vesicles (EVs), have gained attention due to the regulatory effects exerted by their miRNA cargo. Here, we evaluated the impact of preconditioning equine adipose mesenchymal stem cells with TGFβ-1 for short or long periods on the anti-fibrotic properties of secreted extracellular vesicles. MSCs...
Bourebaba L, Bourebaba N, Galuppo L, Marycz K.Nowadays, regenerative medicine techniques are usually based on the application of mesenchymal stem cells (MSCs) for the repair or restoration of injured damaged tissues. However, the effectiveness of autologous therapy is limited as therapeutic potential of MSCs declines due to patient's age, health condition and prolonged in vitro cultivation as a result of decreased growth rate. For that reason, there is an urgent need to develop strategies enabling the in vitro rejuvenation of MSCs prior transplantation in order to enhance their in vivo therapeutic efficiency. In presented study, we attemp...
Bell J, Raidal S, Peters A, Hughes KJ.Management of diarrhoea in horses is usually non-specific and supportive. Faecal microbiota transplantations (FMT) are used to manage dysbiosis in horses with diarrhoea. There are few studies investigating the effects of storage on prepared FMT solutions. This study was an in vitro non-randomised controlled experiment that investigated the effects of FMT solution preparation and storage on the faecal microbiota. Fresh faeces were collected from five healthy adult horses and used for DNA extraction and preparation of FMT. From each FMT, seven aliquots were collected and DNA was extracted immedi...
Duysens J, Graide H, Niesten A, Mouithys-Mickalad A, Deby-Dupont G, Franck T, Ceusters J, Serteyn D.Muscle-derived mesenchymal stromal cells (mdMSCs) hold great promise in regenerative medicine due to their immunomodulatory properties, multipotent differentiation capacity and ease of collection. However, traditional in vitro expansion methods use fetal bovine serum (FBS) and have numerous limitations including ethical concerns, batch-to-batch variability, immunogenicity, xenogenic contamination and regulatory compliance issues. This study investigates the use of 10% equine platelet lysate (ePL) obtained by plasmapheresis as a substitute for FBS in the culture of mdMSCs in innovative 2D and 3...
Yaneselli K, Ávila G, Rossi A, Rial A, Castro S, Estradé MJ, Suárez G, Algorta A.Platelet lysate (PL) is investigated as a potential replacement for fetal bovine serum (FBS) in cell culture. However, there is limited research on its impact on the immune profile of equine mesenchymal stromal cells (eMSCs). This study aimed to evaluate the effects of different PL formulations on the proliferative capacity, multipotentiality, and immune profile of equine adipose tissue-derived MSCs (eAD-MSCs). growth kinetics and trilineage differentiation of eAD-MSCs ( = 7) were assessed under three culture conditions: medium-concentration PL (MPL), high-concentration PL (HPL), and FBS ...
Heidari N, Faragher RGA, Pattison G, Dudhia J, Smith RKW.Tendinopathy is a common age-related disease which causes significant morbidity for both human athletes and performance horses. In the latter, the superficial digital flexor tendon is an excellent model for human tendinopathies because it is a functional homologue of the human Achilles tendon and a primary site of injuries with strong similarities to the human disease. Corticosteroids have been previously used clinically to treat tendinopathic inflammation, but they upregulate the p53-p21 axis with concomitant reductions in cell proliferation and collagen synthesis in human tenocytes. This phe...
Becerro-Rey L, Martín-Cano FE, Fabres Robaina Sancler-Silva Y, Gil MC, Ortega-Ferrusola C, Aparicio IM, Gaitskell-Phillips G, da Silva-Álvarez E....Conservation of equine semen in the liquid state is a central procedure in horse breeding and constitutes the basis of associated reproductive technologies. The intense mitochondrial activity of the stallion spermatozoa increases oxidative stress along the storage period, leading to sperm demise within 24-48 h of storage, particularly when maintained at room temperature. Recently, the relationship between metabolism and oxidative stress has been revealed. The study aimed to extend the period of conservation of equine semen, at room temperature through modification of the metabolites present i...
Li X, Du M, Liu Y, Wang M, Shen Y, Xing J, Zhang L, Zhao Y, Bou G, Bai D, Dugarjaviin M, Xia W.During follicular development, changes in the composition of the follicular fluid are synchronized with the development of oocytes. Our aim was to screen the key factors affecting oocyte maturation and optimize the in vitro culture protocol by understanding the changes of proteins and metabolites in follicular fluid. Follicles are divided into three groups according to their diameter (small follicle fluid (SFF): 10 mm < d < 20 mm; medium follicle fluid (MFF): 20 mm < d < 30 mm; large follicle fluid (LFF): 30 mm < d). Proteins and metabolites from the follicular fluid w...
Sibthorpe PEM, Fitzgerald DM, Sillence MN, de Laat MA.Equine insulin dysregulation (ID) is a significant metabolic problem because the hyperinsulinaemia that develops increases the animal's risk of developing laminitis, a debilitating foot condition. The role of gastrointestinal factors, such as incretin hormones, in the pathogenesis of ID and hyperinsulinaemia in horses is poorly understood, particularly in comparison to other species. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic peptide released from L cells in the gastrointestinal tract and is implicated in metabolic dysfunction in other species. The aim of this study in vitro was to...
Serteyn D, Storms N, Mouithys-Mickalad A, Sandersen C, Niesten A, Duysens J, Graide H, Ceusters J, Franck T.Laminitis in horses is a crippling condition marked by the deterioration of the dermal-epidermal interface, leading to intense lameness and discomfort, often necessitating euthanasia. This study aimed to establish an in vitro model of laminitis using a continuous keratinocyte cell line exposed to anoxia-reoxygenation and an activated neutrophil supernatant. A significant decrease in the keratinocytes' metabolism was noted during the reoxygenation period, indicative of cellular stress. Adding muscle-derived mesenchymal stem/stromal cells during the reoxygenation demonstrated a protective effect...
Philip M, Kal AKK, Subhahar MB, Karatt TK, Graiban FM, Ajeebsanu MM, Joseph M, Jose SV.Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti-inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. Methods: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography-high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. Results: A total of 20 in vivo metabolites wer...
Aguiar LH, Pinto CRF.Sperm capacitation is considered the main factor limiting conventional in vitro fertilization (IVF) in horses. A recent scientific breakthrough in sperm processing for IVF in horses has resulted in embryos and foals being produced; however, various aspects of the IVF process remain to be fully elucidated. Lysine acetylation has been shown to play a role in sperm capacitation in several species and the objective of this study was to detect and evaluate this process in the horse. Ejaculates of two stallions were collected and incubated in different conditions with deacetylase inhibitors to induc...
Vaughn SA, Berghaus LJ, Hart KA.Breed differences exist between horses and ponies in circulating concentrations of several hormones, notably ACTH and insulin. These hormones regulate stress and metabolic responses, but in other species, they also impact leukocyte oxidant responses. The effects of these hormones on equine leukocytes have not been evaluated to date. If equine leukocytes are similarly regulated, breed differences in increased plasma hormone concentrations or altered sensitivity to them at the leukocyte level could result in breed-related differences in oxidant responses or oxidative status. The objective of thi...
Buschmann E, Van Steenkiste G, Duytschaever M, Segers P, Ibrahim L, van Loon G, Decloedt A.Radiofrequency ablation is a promising technique for arrhythmia treatment in horses. Due to the thicker myocardial wall and higher blood flow in horses, it is unknown if conventional radiofrequency settings used in human medicine can be extrapolated to horses. The study aim is to describe the effect of ablation settings on lesion dimensions in equine myocardium. To study species dependent effects, results were compared to swine myocardium. Right ventricular and right and left atrial equine myocardium and right ventricular swine myocardium were suspended in a bath with circulating isotonic sali...
Mazzuchini MP, Lisboa FP, de Castro JI, Alvarenga MA, Segabinazzi LGTM, Canisso IF.Endometritis is the leading cause of subfertility in horses, and it is a clinical problem where historically antibiotics have not always been used with prudent justification. Because of this, alternative therapies to treat endometritis are necessary for the rational use of antibiotics. Objective: To assess the in vitro antimicrobial activity of non-traditional therapies commonly used in clinical practice against microorganisms causing infectious endometritis in mares. Methods: In vitro experiments. Methods: A microdilution technique was performed to determine the minimum inhibitory concentrati...
Lessiak U, Melchert M, Walter I, Kummer S, Nell B, Tschulenk W, Pratscher B.Angiogenesis plays a crucial role in various physiological and pathological conditions. However, research in equine angiogenesis is relative limited, necessitating the development of suitable models. To effectively analyze angiogenesis in-vitro, it is essential to target the specific cells responsible for this process, namely endothelial cells. Human umbilical vein endothelial cells (HUVECs) are one of the most used models for studying angiogenesis in humans. Serving as an equivalent to HUVECs, we present a comprehensive isolation protocol for equine umbilical vein endothelial cells (EqUVECs...
Brito LFC, Felix MR, Linardi RL, Martinez de Andino EV, Balamurugan NS, Hernández-Avilés C, Hinrichs K.Intracytoplasmic sperm injection (ICSI) is a valuable assisted reproduction technology in clinical practice, especially when semen availability is limited. Since the number of sperm required per ICSI cycle is much less than the number of sperm available in a standard straw of frozen semen, refreezing semen at lower sperm concentrations could yield multiple straws for ICSI use. However, there is little data on the effect of sperm refreezing on ICSI outcomes, especially on the effect of extender used for refreezing. The objective of the present study was to evaluate the effect of refreezing exte...
